Antibodies to islet amyloid polypeptide (IAPP) and subunits thereof

ABSTRACT

This invention is directed to antibodies which react with human islet amyloid polypeptide and which do not significantly react with insulin or calcitonin gene-related peptides. Preparations of antibodies are provided which bind to islet amyloid polypeptide (IAPP) which is substantially free of islet amyloid, and when isolated from humans, has the following amino acid sequence in positions 1-37: 
     Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val- His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly- Ser-Asn-Thr-Tyr.

FIELD OF THE INVENTION

The invention was made with the support NIH grant number ROI-DK 36734.The government has certain rights in the invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.7/658,442, filed Feb. 21, 1991, now U.S. Pat. No. 5,112,945 which is acontinuation of U.S. patent application Ser. No. 7/105,267, filed Oct.7, 1987, abandoned.

The invention is concerned with a newly characterized polypeptide andwith antibodies directed against that polypeptide. Potentially thepolypeptide, fragments thereof having the biological activitycorresponding to that of the polypeptide, and proteins containing theamino acid sequence of the polypeptide or of the said fragments thereofcan be employed for therapeutic purposes and for diagnosis of diseasesrelated to the biological function of the polypeptide. Since thepolypeptide has been characterized for the first time in amyloid fibrilsfrom a patient having an insulin-producing pancreatic tumor (insulinoma)this polypeptide will henceforth be called "IAPP", this being anabbreviation of "insulinoma amyloid polypeptide" or "islet amyloidpolypeptide". During the priority year IAPP has also been calleddiabetes-associated peptide (=DAP) (34). Unless otherwise stated, theterm IAPP comprises the polypeptide as such, fragments thereof having acorresponding biological activity, soluble proteins containing the aminoacid sequence of the polypeptide, and furthermore also analogousproteins, polypeptides and fragments from mammals other than humans. Theterm "preparation" is used for the purpose of clearly stating that thepreparations of the invention have been specifically worked up inrespect of IAPP or its homologous antibodies. The invention thus relatesto in vitro preparations and not to the in vivo occurrence of theproteins.

BACKGROUND OF THE INVENTION

Non-insulin-dependent diabetes mellitus (NIDDM) is characterized by animpaired insulin response to elevated glucose levels which, in contrastto insulin-dependent diabetes mellitus (IDDM), is not primarily due toloss of beta-cells even though the total beta-cell mass is moderatelydiminished in NIDDM (1, 2). Despite the apparent multifactorial natureof the pathogenesis of NIDDM, and regardless of whether beta-celldysfunction is primary or secondary, the most substantial and uniformmorphological aspect of this disease with respect to the islets ofLangerhans is the deposition of amyloid. These deposits (isletamyloid=IA) which are exclusively limited to the islets of Langerhansoccur in more than 90% of NIDDM patients and in over 65% of adultdiabetic cats (3-5). IA occurs also in old persons and in aged cats butless frequently and to a lesser extent. IN contrast to the NIDDM cases,no IA deposits have been found in association with IDDM.

The significance of IA has been a matter of discussion ever since thefirst description of the phenomenon in 1990 (6). Although isolated andpurified IA might potentially have served as a useful marker fordetection of an islet cell dysfunction associated with the developmentof NIDDN it has not been possible heretofore to achieve a chemicalanalysis of IA. The principal obstacle to chemical characterization andto amino acid sequencing of the subunits has resided in the difficultyencountered in effectuating solubilization (depolymerization) of the IAfibrils (7). This difficulty of solubilizing IA, which has contributedto the elusive nature of this material, constitutes a direct contrast tothe two major systemic forms of amyloid (i.e. AA or secondary amyloidand AL or primary amyloid) both of which can be depolymerized with 6Mguanidine hydrochloride after suspension in distilled water. Theseproperties of systemic forms of amyloid have permitted purification anddirect chemical analysis of the amyloid fibril protein subunits.

In all instances, amyloid is a pathological deposit of polymerizedsubunits which form beta-pleated sheet fibrils (8). Many different typesof amyloid exist and these may occur systemically or be localized toindividual tissues (9). Each type is characterized by its proteinsubunits, and up to now seven different proteins have been shown to becapable of forming amyloid fibrils in vivo (10-17).

Amyloid in polypeptide hormone producing tissues has been proposed toconsist of corresponding hormones (18, 19). This is best supported forhuman medullary carcinoma of the thyroid where the subunits of theamyloid fibrils formed have been shown, after amino acid sequencing, toconsist of procalcitonin (13). Since amyloid deposits are common ininsulin producing tumors (19) and because there is a close relationshipbetween the amyloid fibrils in islet beta-cells and insulin-producingtumors, it was believed previously that the amyloid in these locationsis derived from insulin or its precursor. The invention is based on ourdiscovery that IA mainly contains a hitherto unknown polypeptide, nownamed IAPP.

In an International Type Search Report compiled by the Swedish PatentOffice during the priority year, the references 26-31 have been cited.References 26-28 deals with unsuccessful attempts to isolate andcharacterize the major component of islet amyloid. Reference 29describes a systemic form of amyloid that is completely distinct fromislet amyloid. See for instance the preceding paragraph. References 30and 31 have been cited as defining the general state of the art and havenot been considered to be of any particular relevance.

SUMMARY OF THE INVENTION

The IAPP preparations of the invention are characterized by containingIAPP while at the same time being substantially free from the amyloidform. If the IAPP derives from a human insulinoma its amino acidsequence is the one set forth in Table 2, especially in respect of theamino acid residues in positions 3-35 or 8-33 (see comments to theTables). The same sequence applies to IAPP isolated from IA taken fromNIDDM patients. As compared with human IAPP the IAPP deriving from othermammals shows minor differences in the amino acid sequence. The term"IAPP preparation according to the invention" comprises also variousderivatives of IAPP. Thus IAPP according to the invention can beprovided with some of the analytically detectable groups that are wellknown in immunochemical assay methods; such as e.g. radioactive,enzymatically active, fluorogenic, chemiluminogenic, biotinyl etc.groups. Also, the IAPP may be attached to any of the various differenttypes of carrier molecules that are well known in immunochemical andimmunosorbent contexts. In the IAPP preparations according to theinvention, the IAPP is usually the principal constituent, i.e. amountsto >50 %, as for instance >90% (w/w) of the proteins from the source ofraw material.

Preparations enriched in IAPP can be produced in that at firstpolymerized IAPP, i.e. IA, is isolated in a manner known per se fromtissues containing such IAPP as e.g. from insulinomas or from islets ofLangerhans of NIDDM patients, whereupon the IA thus obtained isdepolymerized under conditions that will not appreciably hydrolyze thesubunits. As a suitable depolymerizing agent may be made be mentionedconcentrated formic acid. Temperatures may be within the range of 0°-50°C.; but note here that it is always necessary to pay due regard also toall the other working conditions, as e.g. the depolymerizing agentchosen. IAPP may potentially be produced also from cell cultures. Withthe aid of recombinant DNA techniques synthetic DNA and cDNA coding forIAPP may be introduced into microorganisms which will then be made toproduce the peptide. Now that the amino acid sequence of IAPP has becomeknown it is also possible to manufacture IAPP synthetically, in a mannersuch as is known per se for peptide syntheses. In order to be convertedto a pure form the IAPP must be worked up (extracted) from the reactionmixtures (including culture media) which) are obtained in accordancewith any one of the aforesaid methods. For this purpose a large numberof various different biochemical separation procedures may be employed,such as electrophoresis, e.g. isoelectric focusing, centrifugationand/or liquid chromatography. Among these latter may be mentioned highpressure liquid chromatography (HPLC), and gel, affinity and ionexchange chromatography. In particular HPLC will lend itself toproducing a high degree of purity of IAPP.

DETAILED DESCRIPTION OF THE INVENTION

As regards the anti-IAPP antibody preparations of the invention, thesehave to be specific to IAPP and should not react immunochemically withother substances in a manner that would interfere disturbingly with agiven use. The anti-IAPP antibody preparation of the invention has nosignificant reaction with e.g. insulin (native form, A- or B-chainthereof) and calcitonin gene related peptides (CGRPs). Anti-IAPPantibodies may be produced in a manner as is common practice forantibodies, by means of immunizing a suitable animal (e.g. rabbit, rat,mouse etc.) with an IAPP-immunogen, followed by working up the resultantantibodies to obtain a desired purity and form thereof (derivatives,fragments etc.). The production of the anti-IAPP antibodies may beaccomplished by known monoclonal technique. The anti-IAPP antibodies ofthe antibody preparation may thus be present in the form of an antiserumor may be affinity purified, derivatized (e.g. covalently bound to oneof the above-mentioned types of analytically detectable groups, orchemically or physically bound to a phase which is insoluble in aqueousmedia=a so-called "solid" phase) or fragmentized into various anti-IAPPantibody active components, like Fab, Fab' or F(ab')₂.

In order to obtain a high yield and/or quality in the production of ananti-IAPP antibody preparation, the antigen used in the immunizationprotocol or in the selection of the appropriate antibodies in apolyclonal antibody response may be critical. With respect to antigenscomprising only short sequences of IAPP, the sequence of positions 1-6should be avoided. In order to make an efficient selection in apolyclonal antibody response, different combinations of antigens can beused. For instance in monoclonal techniques one can start by selectingIAPP-reacting clones and then in a secondary selection discard thosereacting with CGRPs.

In general terms the production of an antibody preparation according tothe invention (monoclonal as well as polyclonal) is performed in thatcells potentially capable of producing antibodies that possess aspecificity in accordance with the invention are caused to excrete saidantibodies, whereupon the antibodies thus excreted are isolated andpurified so as to remove those antibodies that do not fulfill thespecificity requirements.

Some variant forms of the invention may utilize antibodies or IAPP in asol-called solid-phase-bound form. Binding proteins to solid phases andusing them in e.g. immunological assay methods is prior art technique(20). Examples of solid phases are particulate matrices which arehydrophilic and swellable to form gels but insoluble in water. In manycases such matrices contain OH or NH₂ groups (for example polyamides,polysaccharides, poly(hydroxyalkylacrylates) and correspondingmethacrylates etc.). The antibody or IAPP of the invention may becovalently or adsorptively bound to a water-insoluble matrix.

The protein preparations of the invention may be used for forming, invivo as well as in vitro, so-called ligand-receptor complexes coheringdue to biospecific affinity. The ligand-receptor concept is well knownin patent contexts and comprises such pairs of substances likeenzyme-substrate; hormone-receptor for that hormone; antigen(hapten)-antibody etc. In the present specification and claims, the term"ligand" always refers to IAPP, and the term "receptor" always refers toantibody or the counterpart with which IAPP has to cooperate in vivo inorder that a biological effect be obtained in vertebrates, preferablymammals, such as man.

The ligand-receptor complexes of the invention may be formed

(1) for the purpose of obtaining a biological response in vivo,

(2) in vitro as one of the steps of an immunosorbent process, or

(3) in vitro as one of the steps of an immunochemical assay method.

For forming the complexes in vivo, the IAPP in an amount effective forthe purpose contemplated and incorporated in a suitable sterileformulation is administered to the animal in a site on the animal thatis suitable for the purpose. As vehicles may be mentioned sterileaqueous media such as physiological saline. If a therapeutical treatmentis to be carried out a therapeutically active (=effective) amount isadministered; and in case an immunization is to be carried out theamount of IAPP administered is one that is immunogenically active(=effective), the IAPP being preferably conjugated to an immunogeniccarrier.

For forming the complexes in vitro a preparation according to theinvention is mixed (administered) with (to) a sample containing animmunological counterpart to the agent present in said preparation,under conditions such that the immune complex can be formed. This may bedone in various ways. If several different preparations according to theinvention are employed the exact order is determined by the objects forwhich the immune complex is to be used. The conditions are thosecommonly employed for immune reactions, i.e. aqueous media buffered to apH which will normally be within the range of from pH 3.5 to 9.0,preferably 5.0-8.6. Temperatures are usually maintained in the range offrom +4° to +40° C. Additions may be made of buffers and detergents thatwill not interfere with the immune reaction or its result.

That aspect of the invention which comprises immunosorbent purificationof IAPP involves the binding of anti-IAPP antibodies (e.g.solid-phase-bound) to IAPP, the thus resultant immune complex then beingseparated from the reaction mixture, whereupon in a manner as known perse in connection with immunosorbent purification procedures IAPP can bereleased from the complex and, if required, be subjected to furtherwork-up treatment. In cases where anti-IAPP antibodies are to bepurified, IAPP may be employed in an analogous manner. Temperature andpH are selected as set forth in the preceding paragraph.

The immunochemical assay method of the invention comprises subjectingantibodies directed against IAPP to reaction with the IAPP present in asample to thus form an (IAPP-anti-IAPP) immune complex, the formationand amount of which are measures--qualitative and quantitative,respectively--of the IAPP presence in the sample. For facilitatingdetection and quantification a practice frequently relied on is to addfurther immune reactants or other reactants capable of biospecificallyreacting with constituents of the complex. They may be anti-antibodiesprovided with analytically detectable groups.

Several different immunochemical assay methods are available; theartisan on the basis of his given analyte will decide in each case whichmethod is best for the purpose.

This applies also to IAPP. Among various existent methods may bementioned: competitive (inhibition) and non-competitive methods,precipitation methods, heterogeneous and homogeneous methods, variousmethods named according to the analytically detectable group employed,immunoelectrophoresis, particle agglutination, immunodiffusion andimmunohistochemical methods employing labeled antibodies.

The technical effect of the protein preparations according to thisinvention is demonstrated in the experimental portion, showing that IAPPpreparations may be used for making anti-IAPP antibody preparationswhich in turn may be employed in histochemical assays for IAPP and IA inbiological tissue of e.g. NIDDM patients.

The individual aspects of the invention are defined more specifically inthe attached claims which form an integral part of the specification.

The invention will now be illustrated by means of the underlyingscientific work forming the basis for this invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an elution curve obtained during the purification of subunitsof IA.

EXAMPLES Material and methods A. Purification of subunits of IA(isolation of IAPP)

Amyloid fibrils were isolated from an insulin-producing tumor taken froma 48-year-old man. 2 g of tumor tissue, of which 50% consisted ofamyloid, was homogenized repeatedly in normal saline followed by washingin distilled water until all of the soluble protein had been removed.The resultant pellet was lyophilized, defatted with chloroform-methanol(2:1) and treated with 6M guanidine-HCl, 0.1M Tris HCl, pH 8.0, 0.1MEDTA and 0.1M dithiothreitol. After undissolved material had beencentrifuged and washed in distilled water smears stained with Congo redand examined in polarized light showed that the residual materialconsisted mainly of amyloid. The pellet was lyophilized and treated withformic acid over night at room temperature, involving dissolution ofmost of the material. After centrifugation the supernatant was subjectedto evaporation. 1 mg of the evaporation residue was boiled quickly in0.5 ml of 1% (w/v) sodium lauryl sulfate (=SDS) solution containing0.05M sodium phosphate buffer, pH 7.5. Then 0.2 ml of the reactionmixture was subjected to HPLC on a TSK G2000SW (7.5×600 mm) column(apparatus and column were from LKB, Bromma, Sweden). Elution wasperformed with the phosphate-buffered SDS solution and at a flow rate of0.2 ml/min. The elution curve was taken up at 226 nm with the aid of anLKB 2158 SD wavelength detector equipped with an LKB 2152 integrator(LKB, Bromma, Sweden). The curve is shown as FIG. 1. The column used hasthe capability of separating proteins of molecular weight 500-60,000dalton.

The same procedure was followed in isolating and purifying the subunitsof amyloid from cat Langerhans islets and from an NIDDM patient.

Analysis of IAPP amino acid sequence

Fractions corresponding to the retarded protein peak (B) wereprecipitated with 9 volumes of ethanol at -20° C. The precipitate wasdried, dissolved in 50% trifluoroacetic acid, and applied to a gas phasesequenator (Applied Biosystems, USA). The amino acid residues weredetermined as PTH amino acids directly by HPLC. The percent compositionof amino acids was determined after acid hydrolysis for 24 hours.

Immunohistochemistry. IAPP derivative and its use. Production and use ofanti-IAPP antibody preparation and its use

An undecapeptide corresponding to positions 7-17 of the human insulinomaIAPP and with an extra N-terminal cystine residue was synthesized andconjugated with keyhole limpet hemocyanine by Cambridge ResearchBiochemicals Ltd., Harston, England. The conjugated peptide, dissolvedin 0.1M NaOH and admixed with Freund's complete adjuvant, was injectedsubcutaneously into guinea pigs. Immunization was then continued bymeans of one injection per week, although now with the immunogen inFreund's incomplete adjuvant. Serum was harvested a week after thefourth injection. Human and cat pancreases shown by Congo red stainingto have large amounts of IA, and surgical specimens were fixed in 4%formalin, embedded in paraffin and sectioned. Deparaffinized sectionswere studied by the peroxidaseantiperoxidase method (22) using primaryanti-IAPP antiserum in dilutions of 1:50-1:800. Controls were guinea pigserum and primary antiserum adsorbed with the synthetic peptide (10mg/ml).

By the same technique the presence of IAPP in a dog, mouse, rat, guineapig, hamster and monkey have demonstrated. The technique has also beenapplied for an anti-CGRP antiserum demonstrating a distinct stainingpattern for the latter compared to that of an anti-IAPP antibodypreparation. This indicates that the two antibody preparations havedifferent specificities.

Synthesis of human IAPP₁₋₃₇

Human IAPP:lys-cys-asn-thr-ala-thr-cys-ala-thr-gln-arg-leu-ala-asn-phe-leu-val-his-ser-ser-asn-asn-phe-gly-ala-ile-leu-ser-ser-thr-asn-val-gly-ser-asn-thr-thr

In an attempt to synthesize the complete sequence of human IAPP theprocedure of Mahoney, W. C. (36) has been followed.

The polypeptide is synthesized using a Beckman Model 990B peptidesynthesizer. All amino acid residues are protected at the α-aminoposition with the tert.-butyloxycarbonyl (Boc) group (37). All aminoacids were purchased from Peninsula Laboratories. The synthetic peptidesare deprotected and simultaneously removed from the resin by treatmentwith anhydrous HF, using the SN₂ approach of Tam et al (32).

Peptide purification was accomplished by reversed-phase high-performanceliquid chromatography using volatile buffers as described by Mahoney andHermodson (39), followed by quantification by amino acid analysis. Asmall amount of each peptide (0,5 mg) was modified at the carboxylterminus by suspending the peptide in dry methanol and concentrated HClat ambient temperature for 24 hrs. The resulting peptide methyl esterderivatives were purified by reversed-phase high-performance liquidchromatography, as described above.

Peptide sequencing was accomplished by automated Edman degradation(40-42) using a Beckman Model 890D sequencer, operated using 0.1MQuadrol buffer and a modification of Beckman peptide program 345801.Prior to addition of a sample to the cup 1,8 mg of Polybrene, dissolvedin 50% acetic acid, was applied to the sequencer cup, dried undervacuum, and subjected to three complete cycles of Edman degradation.Subsequently, the sample was applied to the cup and degraded under thedirection of the same program. Samples were prepared for analysis andidentified by high-performance liquid chromatography as previouslydescribed (41, 42).

Amino acid analyses were performed using a Beckman Model 6300 amino acidanalyzer. Spectra were taken using a LKB 4050 Ultraspec which wasinterfaced to a Vacs 11-750 computer. Spectra were taken following a10-min incubation at room temperature upon all reagent additions.

RESULTS

Repeated homogenizations of insulinoma and pancreatic tissue in normalsaline followed by guanidine HCl extraction resulted in both cases inmicroscopically nearly pure amyloid. The amyloid obtained, which wasinsoluble in guanidine-HCl, was dissolved in concentrated formic acid.Lyophilized depolymerized amyloid was soluble in SDS.

Gel chromatography (HPLC) of depolymerized pancreatic IA from humans(insulinoma) as well as cats gave elution profiles having substantiallyjust one single retarded protein peak. (FIG. Peak B.) Fractionscorresponding to this peak were subjected to automated gas phasesequencing of amino acids. One amino acid was released in each step. Theamino acid sequences of the two polypeptides, isolated from insulinomaand from cat Langerhans islets respectively, are shown in Table 1. Theinsulinoma polypeptide was run for 40 cycles of amino acid sequencing,but no amino acid residues could be demonstrated after 37 cycles. Thepercent composition of amino acids as calculated on the basis of theamino acid sequence is in complete agreement with the composition thatcan be calculated after acid hydrolysis (Table 1). No amino acidresidues were found in positions 2 and 36 of the insulinoma polypeptide.Since according to amino acid analysis (Table 2 col. A) the polypeptidecontains cystine (cysteine) and since human CGRP (with which there is acertain degree of partial identity) has cystine bridging position 2 and7, it is highly probable that in the pancreatic tumor polypeptidestudied position 2 is represented by cysteine. Amino acid sequencing ofhuman IAPP was initiated but was discontinued after 18 cycles because bythen no difference from insulinoma IAPP had shown up. The result, takentogether with the similarity in the amino acid composition of thepeptides, reveals them to be identical.

Based on amino acid sequence, the molecular weight of the humanpancreatic tumor peptide is about 3,800-3,900 daltons.

Computer-based comparison with the amino acid sequences of knownproteins, in accordance with the "Search" program (ProteinIdentification Resource, Release 7, Biomedical Research Foundation,Georgetown University Medical Center, Washington, D.C., USA) showspartial identity with the calcitonin gene related peptide (CGRP) (23,24), see Table 3. Although the amino acid sequences of the two peptidesare similar there are also several distinct difference from which it isclear that they are not identical. There is no resemblance topreproinsulin or calcitonin. The comparison has clearly demonstratedthat the amino acid sequence determined by us has not been described forany previously known protein.

The result of N-terminal amino acid sequencing of the cat amyloidpeptide is shown in Table 2. For the amino acid residues of position3-27 the analysis result was conclusive. The sequence thus found isidentical with that determined for the human pancreatic polypeptideexcept for three amino acid substitutions in positions 17, 18 and 23.The incomplete sequence of the cat peptide represents a 44% identitywith the corresponding region of human CGRP.

Using the peroxidase-antiperoxidase technique and primary antiserumdirected against the synthetic peptide segment (positions 7-17 of thehuman tumor peptide), intense immune reactivity was demonstrated inbeta-cells of Langerhans islets from normal human pancreas, and in bothhuman and cat IA. Pancreatic tissues other than islet tissue remainedentirely unstained. Immune reactivity of the islet cells and IA couldnot be obtained if the primary antiserum was first adsorbed with thesynthetic peptide or if the primary antiserum was replaced by normalguinea pig serum.

DISCUSSION

The studies that have been conducive to this invention have been made inthe hope that information about the molecular origin and chemical natureof islet-associated amyloid of both humans and cats will provideimportant insights into the pathogenesis of age-associated diabetesmellitus (DM). IA resembling the human form often occurs in old femalecats afflicted with a form of DM resembling NIDDM (25). Preliminarychemical and immunological studies have shown cat IA to have propertiesvery similar to those of human IA.

The immunohistochemical experiments demonstrate an intense immunereactivity in normal human pancreatic islet cells and in both cat andhuman IA deposits. These results suggest that IAPP is released locallyfrom islet cells.

Human IAPP and cat IAPP are of the same chemical nature, and similaritywith other animals is highly probable. The potential biological andpathobiological significance of this previously unidentifiedneuropeptide-like substance with respect to pancreatic endocrinefunction and pathogenesis of NIDDM is also interesting, especially inview of the fact that we have also demonstrated the occurrence ofamyloid deposits in pancreatic nerves and in ganglia of diabetic cats.These amyloid deposits were shown to have the same histochemical andimmunohistochemical properties as IA and were demonstrated in closeassociation with ganglionic beta-cells. The role of IAPP in islets ofLangerhans is unknown but its partial identity with CGRP stronglyindicates an important regulatory function.

A condition in which IAPP deficiency may conceivably be an importantfactor is insulin-dependent (juvenile) diabetes mellitus (IDDM). Thisdisease is characterized by a more or less total loss of theinsulin-producing beta-cells (1). Since the results hitherto obtainedsuggest that IAPP is synthesized in beta-cells it seems probable thatIDDM also involves IAPP deficiency. Conceivably this deficiency isresponsible for some of the changes seen in IDDM, e.g. kidney lesions orretinopathy. In that case IAPP might perhaps be used therapeutically inIAPP deficiency cases.

TABLE 1

Amino acid compositions of IAPP as purified from amyloid produced byhuman insulinoma (A), by human islets of Langerhans (B), and by felineislets of Langerhans (C). The values set forth are amino acid residuesin % per molecule. Values set forth within parentheses are thecorresponding values calculated on the basis of the amino acid sequenceof IAPP isolated from human insulinoma. Cystine (cysteine) wasdetermined after performic acid oxidation. ND=Not determined.

    ______________________________________                                                 A         B          C                                               ______________________________________                                        Asx        5.5   (6)       4.8      4.0                                       Thr        4.2   (4)       3.6      2.7                                       Ser        5.3   (5)       4.2      3.0                                       Glx        1.9   (1*)      1.8      2.7                                       Pro        0.0   (0)       Trace    1.6                                       Gly        3.4   (3)       4.2      3.2                                       Ala        3.9   (4)       4.2      3.2                                       Cys        1.5   (2)       ND       ND                                        Val        2.1   (2)       2.4      2.2                                       Met        0.0   (0)       0.0      0.0                                       Ile        1.0   (1)       1.2      2.0                                       Leu        2.9   (3)       3.1      4.1                                       Tyr        0.9   (1)       0.9      1.3                                       Phe        1.7   (2)       1.8      1.6                                       His        0.8   (1)       1.2      0.6                                       Lys        1.1   (1)       0.9      2.0                                       Arg        1.3   (1)       1.3      1.8                                       ______________________________________                                         Glx = Glutamine or glutamic acid                                              Asx = Asparagine or aspartic acid                                             *Not yet determined with certainty.   *Not yet determined with certainty.

TABLE 2

Complete amino acid sequence of human insulinoma IAPP compared to a partof cat IAPP. ##STR1##

TABLE 3

Amino acid sequence of human insulinoma IAPP compared to the two humanCGRPs and to the sequenced part of the IAPP from cat IA. The sequencedparts of cat and human IAPP are identical except for positions 17, 18and 23. * not yet determined with certainty ##STR2##

Comments to the tables: The sequences given above differ from thosegiven in the priority application at positions 7 and 34 of human IAPPand 7 of feline IAPP. The correct sequence of human IAPP has beenpublished during the priority year (33, 34).

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We claim:
 1. A purified antibody which reacts with human islet amyloidpolypeptide, which polypeptide is one subunit of islet amyloid and whichis prepared by depolmerizing human islet amyloid, wherein said antibodydoes not significantly react with insulin or with calcitoningene-related peptides selected from the group consisting of human CGRP1and human CGRP2.
 2. A purified antibody which reacts with a polypeptideconsisting essentiallyof:Lys-Cys-Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr,whereinsaid antibody does not significantly react with insulin or withcalcitonin gene-related peptides selected from the group consisting ofhuman CGRP1 and human CGRP2.
 3. A purified antibody which reacts with apolypeptide consisting essentiallyof:Asn-Thr-Ala-Thr-Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly-Ser-Asn,whereinsaid antibody does not react with insulin or with calcitoningene-related peptides selected from the group consisting of human CGRP1and human CGRP2.
 4. A purified antibody which reacts with a polypeptideconsisting essentiallyof:Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Ala-Ile-Leu-Ser-Ser-Thr-Asn-Val-Gly,whereinsaid antibody does not react with insulin or with calcitoningene-related peptides selected from the group consisting of human CGRP1and human CGRP2.
 5. A purified antibody which reacts with a polypeptideconsisting essentiallyof:Cys-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Ile,wherein said antibodydoes not react with insulin or with calcitonin gene-related peptidesselected from the group consisting of human CGRP11 and human CGRP2.
 6. Apreparation of polyclonal antibodies comprising the antibody of claims1, 2, 3, 4 or
 5. 7. A monoclonal antibody preparation comprising theantibody of claims 1, 2, 3, 4 or
 5. 8. A solid phase having attachedthereto the preparation of claim
 6. 9. A solid phase having attachedthereto the preparation of claim
 7. 10. The solid phase of claim 8 whichcomprises a particulate matrix.
 11. The solid phase of claim 9 whichcomprises a particulate matrix.